Matching Items (38)

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Numerical Modeling of Hydrodynamic Flow Focusing in a Microfluidic Device for Time-Resolved Serial Crystallography

Description

Serial femtosecond crystallography (SFX) with an X-ray free-electron laser (XFEL) has enabled the determination of protein structures and protein reaction intermediates in millisecond to microsecond time resolutions. Mix-and-Inject crystallography (MISC)

Serial femtosecond crystallography (SFX) with an X-ray free-electron laser (XFEL) has enabled the determination of protein structures and protein reaction intermediates in millisecond to microsecond time resolutions. Mix-and-Inject crystallography (MISC) at XFELs enables fast mixing in the magnitude of milliseconds in order to achieve desired reaction time points. For these experiments, numerical simulations of a hydrodynamic flow mixer capable of fast mixing by diffusion has been developed using both COMSOL Multiphysics 5.6 and QuickerSims Computational Fluid Dynamics (CFD) Toolbox for MATLAB. These simulation programs were compared by calculations of mixing times and concentration flow profiles. Mixing times in the range of 1-10 ms were calculated in COMSOL under certain flow rate conditions whereas mixing times in the range of 6-15 ms were calculated with QuickerSims. From these mixing times, reaction intermediates can be varied from sub-millisecond to several hundred millisecond time points for a MISC experiment. Explanations for the discrepancies between the two models were attributed to variations in parameter definitions and meshing. Further analysis on the mixing characteristics were investigated by calculating an analytical solution to the convection-diffusion equation for fluid flow in a two-dimensional rectangular channel. The concentration profile along the width of the channel for the analytical solution was compared with the numerical solution obtained with COMSOL and QuickerSims. Upon comparison, it was determined that the diffusion coefficient may not be a significant factor for the disagreement between the two hydrodynamic flow models.

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Date Created
  • 2021-05

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Capacitive Sensor for the Detection of Microdroplets in Serial Femtosecond Crystallography

Description

Microfluidic devices represent a growing technology in the world of analytical chemistry. Serial femtosecond crystallography (SFX) utilizes microfluidic devices to generate droplets of an aqueous buffer containing protein crystals, which

Microfluidic devices represent a growing technology in the world of analytical chemistry. Serial femtosecond crystallography (SFX) utilizes microfluidic devices to generate droplets of an aqueous buffer containing protein crystals, which are then fired out as a jet in the beam of an X-ray free electron laser (XFEL). A crucial part of the device is its method of droplet detection. This project presents a design for a capacitive sensor that uses a unique electrode configuration to detect the difference in capacitance between the aqueous and oil phases. This design was developed using MATLAB and COMSOL Multiphysics simulations and printed using high-resolution 3D printing. Results show that this design can successfully distinguish between the two immiscible liquids, confirming it as a possible detection method in future SFX experiments.

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Date Created
  • 2021-05

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Temperature Measurement In Microfluidic Devics

Description

Microfluidics is an expanding research area for analytical chemistry and the biomedical industry. Microfludic devices have been used for protein and DNA sorting, early detection techniques for cancer and other

Microfluidics is an expanding research area for analytical chemistry and the biomedical industry. Microfludic devices have been used for protein and DNA sorting, early detection techniques for cancer and other disease, and a variety of other analytical techniques. Dielectrophoresis is a technique is often used to control particles within microfluidic devices however the non-uniform electric field can affect the interior of the device. In order to expand the applications of microfluidic devices and to make it easier to work with techniques such as dielectrophoresis, it is essential to understand as much as possible about how the internal environment of the device will affect the sample. A significant part of this is being able to non-invasively determine the temperature inside the microfluidic device in the both the channel and reservoir regions. Several other research group have successfully used temperature sensitive dyes and fluorescence to measure the temperature within microfluidic devices so research began with understanding their techniques and trying to optimize them for the chosen microfluidic channel. Results from calibration and reservoir tests show that there is a linear relationship between the temperature of the channel and the ratio between the dyes Rhodamine 110 and Rhodamine B. Results within the channel showed that the calibration may be difficult to apply directly as absorption from the PDMS continues to be a problem but several coatings can be used to improve the results.

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Date Created
  • 2013-12

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Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction

Description

The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is

The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10–100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ∼4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. This method will also permit an analysis of the dependence of crystal quality on crystal size.

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Date Created
  • 2015-08-19

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Investigating the Relationship Between Astrocytes and Neurons in Alzheimer’s Disease: The Axonal Transport of Amyloid Precursor Protein within Neurons

Description

As Alzheimer’s disease (AD) increases in incidence, there is an increased investigation into the pathogenesis of the disease in hopes of finding a cure to the neurodegenerative disease. The two

As Alzheimer’s disease (AD) increases in incidence, there is an increased investigation into the pathogenesis of the disease in hopes of finding a cure to the neurodegenerative disease. The two key hallmarks of AD consist of amyloid beta plaques and hyperphosphorylated tau fibrillary tangles. Amyloid beta is a peptide that is proteolytically cleaved from the type I transmembrane glycolytic amyloid precursor protein (APP). APP is highly conserved across species, suggesting the importance of APP in healthy brain functioning. However, when APP is cleaved through the amyloidogenic pathway it produces amyloid beta. The trafficking of APP within neurons has been a new endeavor for neurodegenerative disease research, as reduced retrograde trafficking of APP has been hypothesized to increase the likelihood of the amyloidogenic cleavage of APP, resulting in increased amyloid beta presence (Ye et al., 2017). The findings of this study suggest that transport of APP within neurons is significantly inhibited by increased extracellular glutamate concentration. The addition of human primary astrocytes within a human neuron co-culture allowed for significantly increased retrograde transport of APP within neurons, even within high glutamate conditions. These finding enhance the current field of research regarding astrocytes neuroprotective role within the brain, but bring attention to the role that astrocytes have upon regulation of the axonal transport of proteins within neurons.

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Created

Date Created
  • 2019-12

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Insulator based dielectrophoretic trapping of single mammalian cells

Description

This work demonstrated a novel microfluidic device based on direct current (DC) insulator based dielectrophoresis (iDEP) for trapping individual mammalian cells in a microfluidic device. The novel device is also

This work demonstrated a novel microfluidic device based on direct current (DC) insulator based dielectrophoresis (iDEP) for trapping individual mammalian cells in a microfluidic device. The novel device is also applicable for selective trapping of weakly metastatic mammalian breast cancer cells (MCF-7) from mixtures with mammalian Peripheral Blood Mononuclear Cells (PBMC) and highly metastatic mammalian breast cancer cells, MDA-MB-231. The advantage of this approach is the ease of integration of iDEP structures in microfliudic channels using soft lithography, the use of DC electric fields, the addressability of the single cell traps for downstream analysis and the straightforward multiplexing for single cell trapping. These microfluidic devices are targeted for capturing of single cells based on their DEP behavior. The numerical simulations point out the trapping regions in which single cell DEP trapping occurs. This work also demonstrates the cell conductivity values of different cell types, calculated using the single-shell model. Low conductivity buffers are used for trapping experiments. These low conductivity buffers help reduce the Joule heating. Viability of the cells in the buffer system was studied in detail with a population size of approximately 100 cells for each study. The work also demonstrates the development of the parallelized single cell trap device with optimized traps. This device is also capable of being coupled detection of target protein using MALDI-MS.

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Date Created
  • 2013

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Method development in crystallization for femtosecond nanocrystallography

Description

Membrane proteins are a vital part of cellular structure. They are directly involved in many important cellular functions, such as uptake, signaling, respiration, and photosynthesis, among others. Despite their importance,

Membrane proteins are a vital part of cellular structure. They are directly involved in many important cellular functions, such as uptake, signaling, respiration, and photosynthesis, among others. Despite their importance, however, less than 500 unique membrane protein structures have been determined to date. This is due to several difficulties with macromolecular crystallography, primarily the difficulty of growing large, well-ordered protein crystals. Since the first proof of concept for femtosecond nanocrystallography showing that diffraction patterns can be collected on extremely small crystals, thus negating the need to grow larger crystals, there have been many exciting advancements in the field. The technique has been proven to show high spatial resolution, thus making it a viable method for structural biology. However, due to the ultrafast nature of the technique, which allows for a lack of radiation damage in imaging, even more interesting experiments are possible, and the first temporal and spatial images of an undamaged structure could be acquired. This concept was denoted as time-resolved femtosecond nanocrystallography.

This dissertation presents on the first time-resolved data set of Photosystem II where structural changes can actually be seen without radiation damage. In order to accomplish this, new crystallization techniques had to be developed so that enough crystals could be made for the liquid jet to deliver a fully hydrated stream of crystals to the high-powered X-ray source. These changes are still in the preliminary stages due to the slightly lower resolution data obtained, but they are still a promising show of the power of this new technique. With further optimization of crystal growth methods and quality, injection technique, and continued development of data analysis software, it is only a matter of time before the ability to make movies of molecules in motion from X-ray diffraction snapshots in time exists. The work presented here is the first step in that process.

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Created

Date Created
  • 2014

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Blood plasma-based glycan nodes as lung cancer markers and the problem of biospecimen integrity in a multi-site clinical study

Description

Cancer is a major public health challenge and the second leading cause of death in the United States. Large amount of effort has been made to achieve sensitive and specific

Cancer is a major public health challenge and the second leading cause of death in the United States. Large amount of effort has been made to achieve sensitive and specific detection of cancer, and to predict the course of cancer. Glycans are promising avenues toward the diagnosis and prognosis of cancer, because aberrant glycosylation is a prevalent hallmark of diverse types of cancer. A bottom-up “glycan node analysis” approach was employed as a useful tool, which captures most essential glycan features from blood plasma or serum (P/S) specimens and quantifies them as single analytical signals, to a lung cancer set from the Women Epidemiology Lung Cancer (WELCA) study. In addition, developments were performed to simplify a relatively cumbersome step involved in sample preparation of glycan node analysis. Furthermore, as a biomarker discovery research, one crucial concern of the glycan node analysis is to ensure that the specimen integrity has not been compromised for the employed P/S samples. A simple P/S integrity quality assurance assay was applied to the same sample set from WELCA study, which also afford the opportunity to evaluate the effects of different collection sites on sample integrity in a multisite clinical trial.

Here, 208 samples from lung cancer patients and 207 age-matched controls enrolled in the WELCA study were analyzed by glycan node analysis. Glycan features, quantified as single analytical signals, including 2-linked mannose, α2‐6 sialylation, β1‐4 branching, β1‐6 branching, 4-linked GlcNAc, and outer-arm fucosylation, exhibited abilities to distinguish lung cancer cases from controls and predict survival in patients.

To circumvent the laborious preparation steps for permethylation of glycan node analysis, a spin column-free (SCF) glycan permethylation procedure was developed, applicable to both intact glycan analysis or glycan node analysis, with improved or comparable permethylation efficiency relative to some widely-used spin column-based procedures.

Biospecimen integrity of the same set of plasma samples from WELCA study was evaluated by a simple intact protein assay (ΔS-Cysteinylated-Albumin), which quantifies cumulative exposure of P/S to thawed conditions (-30 °C). Notable differences were observed between different groups of samples with various initial handling/storage conditions, as well as among the different collection sites.

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Created

Date Created
  • 2019

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Comparison of SPR and Edge Tracking as a Measure of Binding Kinetics in Whole Cells

Description

Most drugs work by binding to receptors on the cell surface. These receptors can then carry the message into the cell and have a wide array of results. However, studying

Most drugs work by binding to receptors on the cell surface. These receptors can then carry the message into the cell and have a wide array of results. However, studying how fast the binding is can be difficult. Current methods involve extracting the receptor and labeling them, but both these steps have issues. Previous works found that binding on the cell surface is accompanied with a small change in cell size, generally an increase. They have also developed an algorithm that can track these small changes without a label using a simple bright field microscope. Here, this relationship is further explored by comparing edge tracking results to a more widely used method, surface plasmon resonance. The kinetic constants found from the two methods are in agreement. No corrections or manipulations were needed to create agreement. The Bland-Altman plots shows that the error between the two methods is about 0.009 s-1. This is about the same error between cells, making it a non-dominant source of error.

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Date Created
  • 2018

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Targeted proteomics studies: design, development and translation of mass spectrometric immunoassays for diabetes and kidney disease

Description

In an effort to begin validating the large number of discovered candidate biomarkers, proteomics is beginning to shift from shotgun proteomic experiments towards targeted proteomic approaches that provide solutions to

In an effort to begin validating the large number of discovered candidate biomarkers, proteomics is beginning to shift from shotgun proteomic experiments towards targeted proteomic approaches that provide solutions to automation and economic concerns. Such approaches to validate biomarkers necessitate the mass spectrometric analysis of hundreds to thousands of human samples. As this takes place, a serendipitous opportunity has become evident. By the virtue that as one narrows the focus towards "single" protein targets (instead of entire proteomes) using pan-antibody-based enrichment techniques, a discovery science has emerged, so to speak. This is due to the largely unknown context in which "single" proteins exist in blood (i.e. polymorphisms, transcript variants, and posttranslational modifications) and hence, targeted proteomics has applications for established biomarkers. Furthermore, besides protein heterogeneity accounting for interferences with conventional immunometric platforms, it is becoming evident that this formerly hidden dimension of structural information also contains rich-pathobiological information. Consequently, targeted proteomics studies that aim to ascertain a protein's genuine presentation within disease- stratified populations and serve as a stepping-stone within a biomarker translational pipeline are of clinical interest. Roughly 128 million Americans are pre-diabetic, diabetic, and/or have kidney disease and public and private spending for treating these diseases is in the hundreds of billions of dollars. In an effort to create new solutions for the early detection and management of these conditions, described herein is the design, development, and translation of mass spectrometric immunoassays targeted towards diabetes and kidney disease. Population proteomics experiments were performed for the following clinically relevant proteins: insulin, C-peptide, RANTES, and parathyroid hormone. At least thirty-eight protein isoforms were detected. Besides the numerous disease correlations confronted within the disease-stratified cohorts, certain isoforms also appeared to be causally related to the underlying pathophysiology and/or have therapeutic implications. Technical advancements include multiplexed isoform quantification as well a "dual- extraction" methodology for eliminating non-specific proteins while simultaneously validating isoforms. Industrial efforts towards widespread clinical adoption are also described. Consequently, this work lays a foundation for the translation of mass spectrometric immunoassays into the clinical arena and simultaneously presents the most recent advancements concerning the mass spectrometric immunoassay approach.

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Date Created
  • 2011